Urinary tract infections result in approximately 9.6 million doctor visits annually, and 20% of women will develop a urinary tract infection during their lifetime. A majority (70-90%) of urinary tract infections are the result of gram-negative bacteria that produce lipopolysaccharide (LPS), and LPS is the component of the cell wall of gram-negative bacteria responsible for cellular response resulting in pain and inflammation. The mucosa of a variety of organ systems (GI, respiratory, urinary) functions as the first line of innate defense against infection by gram-negative bacteria; however, cellular processes that regulate the response of urothelial cells to LPS remain unknown. Although other investigators have reported that urothelial cells do not express CD14 (the primary cognate cellular receptor for LPS), a recent report described the presence of message for CD14 in human bladder mucosa, and preliminary investigations in our laboratory demonstrated the presence of message and protein in human urothelial cells. These cells also express Toll-Like Receptor 4 (TLR4), which appears to provide the transmembrane pathway for signaling in response to LPS. Urothelial cells have the capacity to produce cytokines which recruit and activate leukocytes. The goal of this research is to test the hypothesis that CD14 and TLR4 play a crucial role in the response of the urothelium to LPS. Using cultures of human urothelial cells and mice with genetic disruption of either CD14 or TLR4, we will investigate the response of the urothelium in vitro and in vivo to LPS in the presence or absence of functional CD14 and TLR4. Further, we will determine whether or not the soluble form of CD14 amplifies the response of urothelial cells to LPS. The proposed experiments will better define participation of these receptors in the response of urothelial cells to LPS in the presence (in vivo) and absence (in vitro) of other components of innate immunity. We anticipate that these studies will provide the foundation for continued investigations to identify additional proteins that may participate in this process and dissect signaling pathways which translate LPS binding by urothelial cells into cellular response. The long-range goal of this research is to identify strategies to minimize pain and harmful cellular effects associated with cystitis due to LPS-producing gram-negative bacteria. [unreadable] [unreadable]